Field Crops Research 91 (2005) 125–130 www.elsevier.com/locate/fcr
Inheritance of resistance to sclerotinia stem rot (Sclerotinia trifoliorum) in faba beans (Vicia faba L.) Anastasios S. Lithourgidisa,*, Dimitrios G. Roupakiasb, Christos A. Damalasc a
b
Department of Agronomy, Aristotle University Farm of Thessaloniki, 570 01 Thermi, Greece Department of Genetics and Plant Breeding, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece c Department of Agronomy, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece Received 8 November 2003; received in revised form 29 May 2004; accepted 16 June 2004
Abstract Stem rot, a fungal disease caused by Sclerotinia trifoliorum Eriks., is often a serious problem in many important forage legumes including faba beans (Vicia faba L.). Understanding the inheritance of resistance to the disease is essential for effective breeding of resistant cultivars. Experiments were conducted to study the inheritance of resistance to stem rot of faba beans. The F1, F2, and the backcross generations of five crosses between four resistant and four susceptible populations (Alto Polycarpe, A-90 Polycarpe, ILB-1814 A-247, A-90 A-244, VT Tanagra) were used. The eight populations were crossed properly in the field, and progenies of F1 and F2, as well as backcross progenies of F1 with each of their parents, were evaluated for resistance to stem rot disease under controlled conditions after artificial inoculation of the plants with carrot root pieces colonized by the fungus. On the assumption that inheritance of stem rot resistance is governed by a single dominant gene, no significant differences were found between the observed and the expected frequencies of resistance for progenies, except for one cross. As the expression of resistance to the disease fits the expected ratios for a single dominant gene model, it is concluded that the inheritance of resistance to sclerotinia stem rot in the evaluated faba bean populations is controlled by a single dominant gene. # 2004 Elsevier B.V. All rights reserved. Keywords: Disease resistance; Genetic analysis; Population
1. Introduction Stem rot is a destructive fungal disease of several economically important forage legumes. The disease, * Corresponding author. Tel.: +30 2310 991789; fax: +30 2310 473556. E-mail address:
[email protected] (A.S. Lithourgidis).
caused by the fungus Sclerotinia trifoliorum Eriks., infects mainly forage legume crops such as alfalfa (Medicago sativa L.), red clover (Trifolium pratense L.), and white clover (Trifolium repens L.), as well as several other legumes (Kohn, 1979). Stem rot disease is also a serious problem for faba beans (Vicia faba L.) in Greece, and frequently it results in a serious and unpredictable yield reduction depending largely on
0378-4290/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.fcr.2004.06.007
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weather conditions (Roupakias, 1983; Lithourgidis et al., 2003). Resistant cultivars are an ideal management tool for reducing yield losses caused by stem rot. Knowledge of the inheritance of resistance to stem rot is, therefore, necessary for the development of effective breeding programs. To our knowledge, the inheritance of stem rot resistance in faba beans has not been investigated. Experimental data on the inheritance of resistance to S. trifoliorum have been reported only in alfalfa (Halimi and Rowe, 1998a, 1998b). These studies indicate that resistance of alfalfa to S. trifoliorum is polygenically controlled. In addition, different modes of genetic control of resistance to Sclerotinia sclerotiorum have been reported in other crops (Abawi et al., 1978; Pirvu et al., 1985; Dickson and Petzoldt, 1996; Kim and Diers, 2000). Data on the genetic basis of resistance to other diseases of faba beans, such as faba bean rust (Uromyces viciae-fabae) and ascochyta blight (Ascochyta fabae), have been reported in the literature. In particular, Conner and Bernier (1982) identified three genes which are responsible for resistance to two rust races in faba bean inbred lines. Furthermore, Rashid et al. (1991) reported that resistance to ascochyta blight of a faba bean inbred line was dominant in crosses with two other inbred lines but recessive in crosses with a third inbred line. Recently, Roma´ n et al. (2003), locating genes associated with A. fabae resistance in faba beans, demonstrated that resistance to ascochyta blight was quantitative. Similarly, Avila et al. (2004) suggested that this resistance should be considered as quantitative trait since several quantitative trait loci have been detected. The objective of this research was to find out the mode of inheritance of the resistance to stem rot caused by the fungus S. trifoliorum in faba beans.
2. Materials and methods Five crosses between resistant and susceptible populations (Alto Polycarpe, A-90 Polycarpe, ILB-1814 A-247, A-90 A-244, VT Tanagra) were carried out at the experimental field of the Aristotle University Farm in Thessaloniki. The populations used in the experiments had been previously ranked as resistant (Alto, A-90, ILB-1814, VT) or
susceptible (Polycarpe, A-247, A-244, Tanagra), as compared with Tanagra which was the most susceptible population (Lithourgidis, 1991). Polycarpe and Tanagra originated from Greece, A-247 from Italy, A-244 from Canada, ILB-1814, Alto and VT from Europe (unknown country), and A-90 obtained from ICARDA. At least 60 plants from each population were pollinated to obtain the F1 seeds of each cross. The F1 plants were selfed to produce F2 populations, and also backcrossed to each of their resistant and susceptible parents. All F1 were grown in isolation until maturity to prevent out-crossing. Parents and progenies were tested for resistance to the disease under a controlled environment using artificial infection. Inoculum preparation and inoculation of faba bean plants were carried out following the procedures described in detail by Lithourgidis et al. (1989, 1991). Carrot root pieces (3 mm 4 mm 5 mm) were autoclaved and placed on potato dextrose agar (PDA) cultures of the fungus which had not yet produced sclerotia. After incubation at 20 8C for 72 h, the colonized root pieces were used as inoculum. Fourweek-old plants were inoculated on the second internode from the top. Immediately after inoculation, the stems were wrapped at the point of inoculation with a piece of wet absorbent cotton to keep the inoculum alive and close to the stem. Subsequently, the inoculated plants were placed in the growth chamber at 19– 21 8C with a 16 h photoperiod of fluorescent light. The plants were sprayed with water three times daily to ensure adequate moisture. After 72 h of incubation, the inoculum was removed and the plants were examined for lesions. Plants with no lesions at all were inoculated again to ensure that there were no escapes during the experimental procedure. For the evaluation of plants, a disease severity scale from 0 to 3 (Lithourgidis et al., 1989) was used as follows: 0 = no visible infection; 1 = early infection indicated by a small lesion; 2 = moderate infection; 3 = severe infection characterized by a large, water-soaked lesion or collapse of the stem. The severity scale was determined after long-term observations on the behavior of the disease in faba beans both in the laboratory and the field. Thus, 7–10 days after inoculation plants with disease severity rating 1 grow normally; plants with disease severity rating 2 may grow or die (percentage mortality about 50%); and plants with disease severity rating 3 normally die (Lithourgidis, 1991). In this
A.S. Lithourgidis et al. / Field Crops Research 91 (2005) 125–130
study, resistance to stem rot was determined 10 days after inoculation: individual plants which survived (grew normally despite the infection) were considered resistant, whereas those which died were considered susceptible. Assuming that resistance to stem rot in faba beans is controlled by a single dominant gene, susceptible plants should have the recessive homozygous genotype (stst), whereas resistant plants should be either homozygous dominant (StSt) or heterozygous (Stst). The frequency of the recessive gene was calculated for each parent population by means of the Hardy–Weinberg law frequencies (Allard, 1960). The expected frequency of the recessive homozygous genotype in the F1 was calculated from the frequency of the recessive gene present in each of the parents; in the backcrosses, it was calculated from the recessive gene frequency in the F1 or reciprocal F1 (RF1) and the frequency of the gene present in the corresponding parent. The expected frequency of the genotypes present in the F2, was calculated from the genotype frequencies present in the F1 (StSt, Stst, stst) after their self fertilization. Subsequently, the observed frequencies of resistance for parents and progenies were compared with the corresponding expected frequencies of resistance using the chi-square (x2) test.
3. Results and discussion Faba bean is a crop with a mixed reproductive system where cross-pollination sometimes exceeds 35% (Bond and Pope, 1974). Thus, it could be con-
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sidered that the populations used in this study are close to Hardy–Weinberg genetic equilibrium. Indeed, in each population it was observed that there was variability among the plants regarding the response to the disease, i.e. within the resistant populations there were susceptible plants and within the susceptible populations there were resistant plants. Thus, the expected ratio (resistant:susceptible) was calculated on the basis of the gene frequencies present in each population. On the assumption that resistance to stem rot in faba beans is controlled by a single dominant gene, the observed frequency of resistant plants does not differ significantly from the expected frequencies in all crosses studied, with the exception of one cross (A90 A-244) (Tables 1–5). Thus, the inheritance of the disease resistance seems to fit the expected ratios for the single dominant gene model. In the cross A-90 A-244, however, the observed frequency of RF1 A90 differed from the expected ones (Table 4). This deviation could be due to cross contamination which may have occurred during the crossing of the two populations. This is supported by the observation that the handling of A-244 plants was particularly difficult due to the small size of their flowers. To our knowledge, no data are available in the literature on the genetic control of resistance to stem rot in faba beans. Data, however, from other crops indicate that the genetic control of resistance to stem rot (either S. sclerotiorum or S. trifoliorum) varies from crop to crop. Thus, the inheritance of resistance to S. sclerotiorum in a Phaseolus vulgaris Phaseolus coccineus cross has been reported to be controlled by a single dominant gene (Abawi et al., 1978).
Table 1 Genetic analysis of sclerotinia stem rot resistance in faba bean populations Alto (resistant) and Polycarpe (susceptible) Genetic material
Alto Polycarpe F1 (Alto Polycarpe) RF1 (Polycarpe Alto)a F2 RF2 F1 Alto RF1 Alto F1 Polycarpe RF1 Polycarpe a
RF1: reciprocal F1.
No. of plants Resistant
Susceptible
43 38 39 30 30 38 24 19 14 34
35 60 41 31 49 47 29 20 22 53
Observed frequency
Expected frequency
x2
P
55.1:44.9 38.8:61.2 48.8:51.2 49.2:50.8 38.0:62.0 44.7:55.3 45.3:54.7 48.8:51.2 38.9:61.1 39.0:61.0
47.6:52.4 47.6:52.4 37.6:62.4 37.6:62.4 51.5:48.5 51.5:48.5 43.4:56.6 43.4:56.6
0.06 0.10 0.0068 2.35 1.54 0.29 0.82 0.79
0.90–0.80 0.80–0.70 0.95–0.90 0.10–0.05 0.30–0.20 0.70–0.50 0.50–0.30 0.50–0.30
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Table 2 Genetic analysis of sclerotinia stem rot resistance in faba bean populations A-90 (resistant) and Polycarpe (susceptible) Genetic material
No. of plants
A-90 Polycarpe F1 (A-90 Polycarpe) RF1 (Polycarpe A-90)a F2 RF2 F1 A-90 RF1 A-90 F1 Polycarpe RF1 Polycarpe a
Resistant
Susceptible
60 15 54 36 33 41 68 39 55 41
14 35 21 18 28 28 33 19 50 36
Observed frequency
Expected frequency
x2
P
81.1:18.9 30.0:70.0 72.0:28.0 66.6:33.4 54.0:46.0 59.4:40.6 67.4:32.6 67.2:32.8 52.4:47.6 53.2:46.8
63.6:36.4 63.6:36.4 50.0:50.0 50.0:50.0 73.9:26.1 73.9:26.1 49.9:50.1 49.9:50.1
2.22 0.39 0.64 3.53 2.19 2.31 0.25 0.44
0.20–0.10 0.70–0.50 0.50–0.30 0.10–0.05 0.20–0.10 0.20–0.10 0.70–0.50 0.70–0.50
RF1: reciprocal F1.
Table 3 Genetic analysis of sclerotinia stem rot resistance in faba bean populations ILB-1814 (resistant) and A-247 (susceptible) Genetic material
No. of plants
ILB-1814 A-247 F1 (ILB-1814 A-247) RF1 (A-247 ILB-1814)a F2 RF2 F1 ILB-1814 RF1 ILB-1814 F1 A-247 RF1 A-247 a
Resistant
Susceptible
28 19 10 32 26 30 15 16 15 14
5 34 3 14 18 22 3 4 15 13
Observed frequency
Expected frequency
x2
P
74.9:15.1 35.9:64.1 77.0:23.0 69.6:30.4 59.1:40.9 57.7:42.3 83.3:16.7 75.0:25.0 50.0:50.0 51.9:48.1
68.8:31.2 68.8:31.2 54.6:45.4 54.6:45.4 78.3:21.7 78.3:21.7 53.0:47.0 53.0:47.0
3.13 0.03 0.82 0.39 1.47 0.64 0.36 0.049
0.10–0.05 0.70–0.50 0.50–0.30 0.70–0.50 0.30–0.20 0.50–0.30 0.70–0.50 0.90–0.80
RF1: reciprocal F1.
Further, in two sunflower (Helianthus annuus L.) cultivars, resistance was found to be controlled by a single recessive gene (Pirvu et al., 1985) and by a major recessive gene plus modifiers in cabbage (Bras-
sica oleracea L.) (Dickson and Petzoldt, 1996), while for two soybean (Glycine max L.) cultivars it has been reported that resistance to S. sclerotiorum is controlled at least by two recessive genes (Hoffman et al., 1999).
Table 4 Genetic analysis of sclerotinia stem rot resistance in faba bean populations A-90 (resistant) and A-244 (susceptible) Genetic material
No. of plants Resistant
Susceptible
A-90 A-244 F1 (A-90 A-244) RF1 (A-244 A-90)a F2 RF2 F1 A-90 RF1 A-90 F1 A-244 RF1 A-244
60 10 26 28 22 18 12 11 9 13
14 34 16 24 24 18 7 7 12 22
a
RF1: reciprocal F1.
Observed frequency
Expected frequency
x2
P
81.1:18.9 22.7:77.3 61.9:38.1 53.9:46.1 47.8:52.2 50.0:50.0 63.2:36.8 61.1:38.9 42.9:57.1 37.2:62.8
61.8:38.2 61.8:38.2 48.0:52.0 48.0:52.0 73.1:26.9 73.1:26.9 45.7:54.3 45.7:54.3
0.0004 2.64 0.0015 0.16 4.83 7.32 0.31 2.91
0.99–0.95 0.20–0.10 0.95–0.90 0.70–0.50 0.05–0.02 0.01–0.001 0.70–0.50 0.10–0.05
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Table 5 Genetic analysis of sclerotinia stem rot resistance in faba bean populations VT (resistant) and Tanagra (susceptible) Genetic material
VT Tanagra F1 (VT Tanagra) RF1 (Tanagra VT)a F2 RF2 F1 VT RF1 VT F1 Tanagra RF1 Tanagra a
No. of plants Resistant
Susceptible
27 18 22 25 67 7 8 9 6 –
15 41 23 34 82 10 6 10 12 –
Observed frequency
Expected frequency
x2
P
64.3:35.7 30.5:69.5 48.9:51.1 42.4:57.6 45.0:55.0 41.2:58.8 57.1:42.9 47.4:52.6 33.3:66.7 –
50.2:49.8 50.2:49.8 39.4:60.6 39.4:60.6 58.0:42.0 58.0:42.0 41.0:59.0 41.0:59.0
0.067 2.44 1.30 0.14 0.033 4.61 2.45 –
0.80–0.70 0.30–0.10 0.30–0.20 0.80–0.70 0.90–0.80 0.05–0.02 0.20–0.10 –
RF1: reciprocal F1.
In contrast, Dickson et al. (1982) and Fuller et al. (1984) reported that inheritance of resistance to S. sclerotiorum in dry beans was quantitative. Polygenic control of resistance to stem rot has also been reported in sunflower (Robert et al., 1987; Castan˜ o et al., 2001) and soybean (Kim and Diers, 2000). Similarly, in alfalfa, the resistance to S. trifoliorum has been reported to be governed by two qualitatively different systems, with different genes probably involved in each system, suggesting polygenic control of resistance (Halimi and Rowe, 1998a, 1998b). The genetic analysis of our data suggests that the inheritance of resistance to stem rot in the faba bean populations studied is controlled by a single dominant gene. Similarly, Kohpina et al. (2000) found that resistance to ascochyta blight in a faba bean population was due to a single dominant gene, whereas in another faba bean population resistance to the disease was found to be recessive and multigenic. Furthermore, Stoddard and Herath (2001) reported that additive gene action was very important in determining the response to rust disease resistance in faba beans populations and diallel F1 hybrids. For a complete study of the inheritance of sclerotinia stem rot resistance in faba beans, inbred lines should be developed from both the susceptible and the resistant populations and properly crossed. The present data, apart from being a starting point for further investigation of the genetic control of stem rot resistance in faba beans, could be useful for the development of effective breeding programs that might lead to faba bean cultivars resistant to this destructive disease.
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